This invention relates to viral vectors and methods for their use, especially for example for transducing cells, for example malignant cells of hemopoietic lineage, and for inducing the expression of foreign genetic material in such cells. The invention also relates to pharmaceutical compositions based on such viral vectors, to the production of cells infected with such viral vectors, to pharmaceutical preparations based on such cells, and to their use for administration to humans and to non-human animals in order to achieve expression of foreign genetic material in vivo. Methods according to the invention can be used for example in cancer immunotherapy.
Recombinant viral vectors are among several known agents available for the introduction of foreign genes into cells so that they can be expressed as protein. A central element is the target gene itself under the control of a suitable promoter sequence that can function in the cell to be transduced. Known techniques include non-viral methods, such as simple addition of the target gene construct as free DNA; incubation with complexes of target DNA and specific proteins designed for uptake of the DNA into the target cell; and incubation with target DNA encapsulated for example in liposomes or other lipid-based transfection agents.
A further option is the use of recombinant virus vectors engineered to contain the required target gene, and able to infect the target cells and hence carry into the cell the target gene in a form that can be expressed. A number of different viruses has been used for this purpose including retroviruses, adenoviruses, and adeno-associated viruses.
Specification EP 0 176 170 (Institut Merieux: B Roizman) describes foreign genes inserted into a herpes simplex viral genome under the control of promoter-regulatory regions of the genome, thus providing a vector for the expression of the foreign gene. DNA constructs, plasmid vectors containing the constructs useful for expression of the foreign gene, recombinant viruses produced with the vector, and associated methods are disclosed.
Specification EP 0 448 650 (General Hospital Corporation: Al Geller, XO Breakefield) describes herpes simplex virus type 1 expression vectors capable of infecting and being propagated in a non-mitotic cell, and for use in treatment of neurological diseases, and to produce animal and in vitro models of such diseases.
Recombinant viruses are known in particular for use in (e.g. corrective) gene therapy applied to gene deficiency conditions.
Examples of genes used or proposed to be used in corrective gene therapy include: the gene for human adenosine deaminase (ADA), as mentioned in for example WO 92/10564 (KW Culver et al: US Secretary for Commerce and Cellco Inc), and WO 89/12109 and EP 0 420 911 (IH Pastan et al); the cystic fibrosis gene and variants described in WO 91/02796 (L-C Tsui et al: HSC Research and University of Michigan), in WO 92/05273 (F S Collins and J M Wilson: University of Michigan) and in WO 94/12649 (RJ Gregory et al: Genzyme Corp).
The prior art of malignant tumor treatment includes studies that have highlighted the potential for therapeutic vaccination against tumors using autologous material derived from a patient""s own tumor. The general theory behind this approach is that tumor cells may express one or more proteins or other biological macromolecules that are distinct from normal healthy cells, and which might therefore be used to target an immune response to recognise and destroy the tumor cells.
These tumor targets may be present ubiquitously in tumors of a certain type. A good example of this in cervical cancer, where the great majority of tumors express the human papillomavirus E6 and E7 proteins. In this case the tumor target is not a self protein, and hence its potential as a unique tumor-specific marker for cancer immunotherapy is clear.
There is increasing evidence that certain self proteins can also be used as tumor target antigens. This is based on the observation that they are expressed consistently in tumor cells, but not in normal healthy cells. Examples of these include the MAGE family of proteins. It is expected that more self proteins useful as tumor targets remain to be identified.
Tumor associated antigens and their role in the immunobiology of certain cancers are discussed for example by P van der Bruggen et al, in Current Opinion in Immunology, 4(5) (1992) 608-612. Other such antigens, of the MAGE series, are identified in T. Boon, Adv Cancer Res 58 (1992) pp 177-210, and MZ2-E and other related tumor antigens are identified in P. van der Bruggen et al, Science 254 (1991) 1643-1647; tumor-associated mucins are mentioned in PO Livingston, in current Opinion in Immunology 4 (5) (1992) pp 624-629; e.g. MUC1 as mentioned in J Burchell et al, Int J Cancer 44 (1989) pp 691-696.
Although some potentially useful tumor-specific markers have thus been identified and characterised, the search for new and perhaps more specific markers is laborious and time-consuming.
An experimental intracranial murine melanoma has been described as treated with a neuroattenuated HSV1 mutant 1716 (BP Randazzo et al, Virology 211 (1995) pp 94-101), where the replication of the mutant appeared to be restricted to tumor cells and not to occur on surrounding brain tissue.
Administration to mammals of cytokines as such (i.e. as protein) has been tried, but is often poorly tolerated by the host and is frequently associated with a number of side-effects including nausea, bone pain and fever. (A Mire-Sluis, TIBTech vol. 11 (1993); MS Moore, in Ann Rev Immunol 9 (1991) 159-91). These problems are exacerbated by the dose levels often required to maintain effective plasma concentrations.
It is known to modify live virus vectors to contain genes encoding a cytokine or a tumor antigen. Virus vectors have been proposed for use in cancer immunotherapy to provide a means for enhancing tumor immunoresponsiveness. Specification WO 86/07610 (Transgene: MP Kieny et al) discloses expression of human IL-2 in mammalian cells by means of a recombinant poxvirus comprising all or part of a DNA sequence coding for a human IL-2 protein. Specification EP 0 259 212 (Transgene SA: R Lathe et al) discloses viral vectors of the pox, adeno or herpes types, for controlling tumors, containing a heterologous DNA sequence coding for at least the essential regions of a tumor-specific protein. Specification WO 88/00971 (CSIRO, Australian National University: Ramshaw et al) discloses recombinant vaccine comprising a pox, herpes or adeno virus vaccine vector, especially vaccinia, including a nucleotide sequence expressing at least part of an antigenic polypeptide and a second sequence expressing at least part of a lymphokine (interleukin 1, 2, 3 or 4, or gamma interferon) which increases immune response to the antigenic polypeptide; and specification WO 94/16716 (E Paoletti et al: Virogenetics Corp.) describes attenuated recombinant vaccinia viruses containing DNA coding for a cytokine or a tumor antigen, e.g. for use in cancer therapy.
It has been proposed to use GMCSF-transduced tumor cells as a therapeutic vaccine against renal cancer. The protocols for corresponding trials involve removal of tumor material from the patients, and then transduction with the appropriate immunomodulator gene. The engineered cells are then to be re-introduced into the patient to stimulate a beneficial immune response.
Vectors based on herpesvirus saimiri, a virus of non-human primates, have been described as leading to gene expression in human lymphoid cells (B Fleckenstein and R Grassmann, Gene 102(2) (1991), pp 265-9). However, it has been considered undesirable to use such vectors in a clinical setting.
Although it is therefore known to introduce immunomodulatory and other genes into cells such as certain kinds of tumor cells, existing methods of achieving this are considered by the present inventors to have limitations, whether the difficulties are due to low quantitative amounts of transduction, to complexity, or to undesirable side-effects of the systems employed.
The present inventors consider that it has been difficult up to now to introduce genes into a number of kinds of cells, e.g. tumor cells of hemopoietic lineage, such as leukaemias, or to do this efficiently, e.g. for purposes of corrective gene therapy or cancer immunotherapy.
For the transfer of genes to such cells as hemopoietic progenitor cells, retroviral vectors have been the most widely tried vectors up to the present. It appears that these vectors however do not integrate and are not expressed in nondividing cells, and this limits their value e.g. when used with for example hemopoietic stem cells (HSCs) or primary cells from human hemopoietic malignancies as targets for gene transfer and expression. In order to overcome this limitation, culture of target cells, e.g. HSCs, with hemopoietic growth factors such as cytokines has been tried, with a view to induce the HSCs into cycle and increase the efficiency of retrovirus-mediated gene transfer to these target cells, but unfortunately the cytokines in the culture media appear to have induced differentation with loss of the desired self-renewal capacity of the cells.
Thus, adeno-associated viral vectors have been proposed for use instead of retroviral vectors, but it has appeared that the efficiency of integration of such vectors is low.
Also, the present inventors consider, on the basis of recent experience with adenoviral vectors, that these have limitations. Thus, while they can infect approximately 50% of hemopoietic cells under certain conditions, nevertheless gene expression is often delayed for several days. It has also been found in certain tests that transduction of a heterologous gene into acute leukaemia cells by a recombinant adenovirus vector or a retrovirus vector led to either negligible or at best about 3% transduction yield, and that thus there can be a problem of efficiency of transduction yield with such vectors.
The present inventors consider that the prior art leaves it still desirable to provide further viral vectors and processes for their use in transforming human and animal cells. In particular, it remains desirable to provide materials and methods to produce gene transfer to human and non-human animal cells with useful rapidity. Also desirable is to provide materials and methods to produce gene transfer with useful efficiency. Also desirable is the provision of materials and methods to produce gene transfer with applicability to a useful range of target cell types, usefully including for example non-dividing cells.
According to an aspect of the invention described herein, target cells for transduction by herpesviral vectors can be chosen for example from among cells of hemopoietic lineages; from lymphoid or myeloid cells, from stem cells or CD34+ cells, e.g. cell preparations containing such cells, as for example obtained or prepared in connection with bone-marrow transplantation; or cells of neuroectodermal origin, especially malignant such cells, and transduced with viral vectors as described herein. In this use, it has been found that certain methods and procedures according to examples of the invention can lead to surprisingly high transduction efficiency.
In one aspect the present invention aims to provide materials and methods to facilitate the use of tumor cells as immunogens and vaccines. In a further aspect the invention aims to facilitate the transduction of cells of hemopoietic lineage and provide useful compositions and procedures based thereon.
The present invention also aims to provide means for creating immunogens and therapeutic vaccines that can be used to induce immune responses against tumor-specific antigens, e.g. in patients with pre-existing tumors.
The invention is particularly applicable for example for gene transfer into hemopoietic cells such as lymphoid cells, that are nonpermissive for expression of late lytic genes of herpesvirus such as herpes simplex virus.
According to an aspect of the invention there is provided a process of treating a human or non-human animal cell to introduce heterologous genetic material, e.g. material comprising a heterologous gene, into said cell, e.g. to express said genetic material in said cell, comprising the steps of (a) providing a recombinant herpesviral vector which is an attenuated or replication-defective and non-transforming mutant herpesvirus, and which carries heterologous genetic material, e.g. a gene encoding a heterologous protein, and (b) transducing human or non-human animal cells selected from: hemopoietic cells, malignant cells related to blood cells, and malignant or non-malignant CD34+ cells; by contacting said cells with said virus vector to transduce said cells. In embodiments of the invention described below said genetic material is then expressed in said cell. Transduction takes place by infection of the live target cell by the viral vector in per-se known manner.
Such a process can for example comprise treating a human or non-human animal cell to introduce heterologous genetic material into said cell to render said cell more highly immunogenic, comprising the steps of (a) providing a recombinant herpesviral vector which is an attenuated or replication-defective and non-transforming mutant herpesvirus, and which carries e.g. a gene encoding a heterologous immunomodulatory protein selected from cytokines and immunological co-stimulatory molecules and chemo-attractants, and (b) transducing malignant or non-malignant human or non-human animal cells, which can be selected for example from: malignant cells related to blood cells, hemopoietic cells, malignant or non-malignant CD34+ cells, by contacting said cells with said virus vector to transduce said cells and render said cells more highly immunogenic.
Pharmaceutical preparations provided and used according to certain embodiments of the invention, for use in transducing human or non-human animal cells selected from: hemopoietic cells; malignant cells related to blood cells; and malignant or non-malignant CD34+ cells; can comprise a recombinant herpesviral vector which is an attenuated or replication-defective and non-transforming mutant herpesvirus, and which carries heterologous genetic material, e.g. a gene encoding a heterologous protein.
Pharmaceutical preparations provided and used according to certain embodiments of the invention can comprise human or non-human animal cells selected from: hemopoietic cells; malignant cells related to blood cells; and malignant or non-malignant CD34+ cells; said cells having been infected with a recombinant herpesviral vector which is an attenuated or replication-defective and non-transforming mutant herpesvirus, and which carries e.g. a gene encoding a heterologous protein.
Also within the invention is a process of treating a subject which is a human subject or a non-human animal subject in order to achieve expression of a foreign gene in vivo, comprising administering to said subject a pharmaceutical composition of the kinds mentioned above and described herein; and a process of treating a subject which is a human subject or a non-human animal subject in order to elicit an immune response, which comprises administering to said subject a pharmaceutical composition of the kinds mentioned above and described herein.
An aspect of the invention concerns provision and use of a recombinant herpesvirus vector, e.g. based on a non-transforming herpesvirus, carrying a gene encoding a protein, e.g. an immunomodulatory protein, or a protein useful for expression in connection with gene therapy: also provided by the invention is its use in transducing cells to render them more highly immunogenic; among the cells that can usefully be treated in this way are for example malignant cells of human and non-human animals, especially for example malignant cells related to blood cells, e.g. leukaemic cells, or hemopoietic cells, including CD34+ cells, whether malignant or non-malignant. Thus suitable cells for treatment include for example hemopoietic progenitor cells such as healthy CD34+ cells, which when transduced with herpesvirus vectors carrying a heterologous gene that it is desired to express in the treated cell, can carry a high copy number of the heterologous gene, enabling homologous recombination with the genome of the treated cell without the need for an integrase.
Among the applications of embodiments of the present invention is the modification of malignant hemopoietic cells by the transfer of genes to generate tumor immunogens. Among the substances that can usefully be generated in a modified cell to function as a tumor immunogen are GM-CSF and interleukin 2. For example, it has been reported that interleukin 2 production by tumor cells bypasses T helper function in the generation of an antitumor response (ER Fearon et al, Cell 60 (1990) pp 397 et seq), and it has been reported in the case of murine GM-CSF (G Dranoff et al, Proc Nat Acad Sci USA 90 (1993) pp 3539 et seq.) that vaccination with irradiated tumor cells engineered to secrete GM-CSF stimulates potent, specific and long lasting anti-tumor immunity.
Thus, according to embodiments of the invention, a recombinant herpesvirus, for example a recombinant HSV, can be used as a vector for transduction of (for example) leukaemia cells so as to produce expression of inserted genetic material, e.g. a gene encoding an immunomodulatory protein or other protein relevant to cancer immunotherapy or gene therapy, in such cells.
In particular examples of the invention, a recombinant herpes simplex virus, whether HSV1 or HSV2, engineered to contain a heterologous gene as part of its genome, can be used to deliver the gene with good efficiency to leukaemia cells, to evoke effective expression of the heterologous gene within the tumor cells, and the transduced cells can then be used for example as a cellular immunogen such as a vaccine for cancer immunotherapy, and thereby, among other effects, mediate immune effects on tumor cells other than cells infected with the virus vector. Thus the invention also provides useful methods for gene transduction of leukaemia cells among others.
Also provided according to certain embodiments of the invention are methods of using a recombinant herpesvirus such as HSV, e.g. a replication-defective herpesvirus such as replication-defective HSV, whether HSV1 or HSV2, for transduction of various cell types based on cells of hemopoietic lineage, and other cell types, e.g. neuroblastomas, e.g. to introduce immunomodulatory genes, or other genes for the purpose of gene therapy or cancer immunotherapy, into such cells.
It has also been found that transduction into leukemia cells using an example of a HSV-based recombinant vector can be achieved successfully using fresh tumor cells. Thus, tumor cells, which can be cells that (prior to transduction) either have not been incubated at all under cell culture conditions, or else have not been incubated for more than a few hours (e.g. not more than about 2 or up to 4 hours, or not incubated as long as overnight), e.g. freshly-sampled tumor cells, can be exposed to a recombinant herpesvirus vector as mentioned herein carrying suitable genetic material. This can be genetic material that is not being expressed, or is not being substantially expressed, by the tumor cells, e.g. genetic material encoding an immunomodulatory protein such as for example GM-CSF, thereby to infect the cells with the recombinant herpesvirus vector; and the resulting infected cells can be used for example either for reinfusion into the subject from whom the parent cells were obtained, or for reaction with leukocytes in vitro.
For example, freshly sampled human leukaemia cells can be exposed to a virus vector carrying a gene encoding human GM-CSF or inter alia one of the other immunomodulatory proteins mentioned herein, and reinfused into the patient as an immunogenic cell preparation, e.g. using some or all of the procedural steps mentioned below, with a useful extent of transduction of the cells. By contrast, previously, using a corresponding retrovirus vector, it has proved necessary to culture the tumor cells in vitro for some days before they could be transduced usefully; e.g. in order to drive cells into cell division and render them susceptible to retroviral transduction.
This can be a useful advantage of recombinant herpesvirus vectors as described herein, since it can reduce the need for laboratory manipulation of the tumor cells, can be more rapid, with more efficient cell transduction, and can present a more viable clinical treatment option.
Cytotoxic T-cells can be activated and/or expanded, e.g. in vitro, e.g. for purposes of cancer immunotherapy, by the use of virally-transduced presenting or target cells, e.g. especially target cells of hemopoietic lineage, CD34+ cells, where the virus used for transduction is a vector as described herein carrying a gene encoding an antigen relevant to the desired therapy, e.g. an antigen encoded by EBV or HPV, and in addition, if desired, encoding an immunomodulatory protein as mentioned herein. An example of such use is the case of donor-cell tumors in transplant patients where the tumor cells express EBV or HPV antigens: donor T-lymphocytes can be activated and expanded in relation to target cells, e.g. of types as mentioned above, expressing EBV or HPV antigens as a result of transduction by viral vectors as described herein carrying corresponding heterologous genes, e.g. HPV E6 or E7 genes. Recombinant herpesvirus as mentioned herein can also transduce other tumor cell types, such as neuroblastoma cells, with good efficiency.
The recombinant herpesvirus used as a vector according to this invention can contain a gene encoding an immunomodulatory protein, or other protein relevant to cancer immunotherapy or gene therapy.
Genes encoding any of several immunomodulatory proteins can be used in this way to render tumor cells immunogenic, in humans and non-human animals. The resulting immune responses can be used in prevention and treatment of tumor growth.
Immunomodulators are molecules that can enhance or repress immunological responses. They include cytokines (soluble glycoproteins which initiate or enhance activation, growth and differentiation of immune system cells), co-stimulatory molecules (structures present on the surface of cells within the body that interact with immune cells to help stimulate immune responses) and (immunological) chemo-attractant molecules which serve to attract immune cells to sites of immune or inflammatory activity, e.g. at which antigens can be presented. xe2x80x9cImmunomodulatingxe2x80x9d or xe2x80x9cimmunomodulatoryxe2x80x9d protein, as referred to herein, includes one or more proteins which can enhance a host""s immune response, e.g. to a mutant virus, or to an antigen such as an immunogen from a pathogen or source exogenous to the virus, or to a tumor associated antigen, which can for example be produced by the mutant virus. The immunomodulating proteins are not those presently used as immunogens in themselves. The immunodulating proteins for which encoding nucleotide sequences are expressibly carried by viruses as described herein can for example usefully have sequences native to the species which is to receive vaccination by the recombinant viruses, or which is otherwise to receive cells transduced with the recombinant viruses, e.g. it is recommended to use an immunomodulating protein of substantially human sequence for transducing a cell preparation to be used as a human immunogen or vaccine, or to be used otherwise in connection with humans.
Any hazards associated with expression of such proteins in a fully replicating virus are eliminated where the virus is a replication defective mutant. In certain embodiments, the proteins can be selected to enhance the effect of the mutant virus as an immunogen or vaccine in the context in which it is employed.
Examples of useful immunomodulating proteins include cytokines for example interleukins 1 to 15 (IL1 to IL15), interferons alpha, beta or gamma, tumor necrosis factor (TNF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), chemokines such as neutrophil activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), RANTES, macrophage inflammatory peptides MIP-1 a and MIP-1 b, complement components and their receptors, accessory molecules such as one of the B7 family of T cell co-stimulators such as B7.1 or B7.2, ICAM-1, 2 or 3, OX40 ligand and cytokine receptors. Where nucleotide sequences encoding more than one immunomodulating protein are inserted, they may comprise more than one cytokine or may be a combination of cytokine(s) and accessory molecule(s). Many further kinds of immunomodulatory proteins and genes can be useful in this invention.
Examples of particularly useful immunomodulatory proteins include GMCSF; IL2; IL4; IL7; IL12; B7.1; TNF-alpha; interferon gamma; CD40L; and lymphotactin.
The genetic material encoding an immunomodulatory protein can be carried in the mutant viral genome as an expressible open reading frame encoding a hybrid or fusion protein which comprises a polypeptide region having homology to and functionality of an immunomodulatory protein, linked to a polypeptide region having another homology and optionally another functionality. For example, the immunomodulatory protein can be, comprise, or correspond in functionality to the gp34 protein identified as a binding partner to human OX-40 (see W Godfrey et al, J Exp Med 180(2) 1994 pp 757-762, and references cited therein, including S Miura et al, Mol Cell Biol 11(3) 1991, pp 1313-1325). The version of this protein functionality that can be encoded in the mutant viral genome can correspond to the natural gp34 sequence itself, or to a fragment thereof, or to a hybrid expression product e.g. based on the (C-terminal) extracellular (binding) domain of gp34 fused to another protein, e.g. to the constant region of an immunoglobulin heavy chain such as human IgG1, e.g. with the extracellular domain of gp34 (a type 2 membrane protein) fused at its N-terminal to the C-terminal of the immunoglobulin constant domain.
Others of the immunomodulatory proteins can also be carried and expressed in corresponding or other derivative and hybrid forms. It is also understood that mutations of the aminoacid sequences of such immunomodulatory proteins can be incorporated. Included here are proteins having mutated sequences such that they remain homologous, e.g. in sequence, function, and antigenic character, with a protein having the corresponding parent sequence. Such mutations can preferably for example be mutations involving conservative aminoacid changes, e.g. changes between aminoacids of broadly similar molecular properties. For example, interchanges within the aliphatic group alanine, valine, leucine and isoleucine can be considered as conservative. Sometimes substitution of glycine for one of these can also be considered conservative. Interchanges within the aliphatic group aspartate and glutamate can also be considered as conservative. Interchanges within the amide group asparagine and glutamine can also be considered as conservative. Interchanges within the hydroxy group serine and threonine can also be considered as conservative. Interchanges within the aromatic group phenylalanine, tyrosine and tryptophan can also be considered as conservative. Interchanges within the basic group lysine, arginine and histidine can also be considered conservative. Interchanges within the sulphur-containing group methionine and cysteine can also be considered conservative. Sometimes substitution within the group methionine and leucine can also be considered conservative. Preferred conservative substitution groups are aspartate-glutamate; asparagine-glutamine; valine-leucine-isoleucine; alanine-valine; phenylalanine-tyrosine; and lysine-arginine. In other respects, mutated sequences can comprise insertion and/or deletions.
Particular useful examples of derivative and hybrid forms include proteins with sequences having deleted therefrom any of: a transmembrane segment, an intracellular sequence portion, an N-terminal or C-terminal sequence, e.g. a sequence of from 1-5 aminoacids upwards; and/or sequences having added thereto any of e.g. an N-terminal or C-terminal sequence, e.g. a sequence of from 1-5 aminoacids upwards, or a further functional sequence e.g. as described above.
Suitably the immunomodulating protein can comprise a cytokine, e.g. granulocyte macrophage colony stimulating factor (GM-CSF). Murine GM-CSF gene, for example, encodes a polypeptide of 141 amino acids, the mature secreted glycoprotein having a molecular weight of between 14k-30k daltons depending on the degree of glycosylation. GM-CSF is a member of the hemopoietic growth factor family and was first defined and identified by its ability to stimulate in vitro colony formation in hemopoietic progenitors. GM-CSF is a potent activator of neutrophils, eosinophils and macrophage-monocyte function, enhancing migration, phagocytosis, major histocompatibility complex (MHC) expression, and initiating a cascade of bioactive molecules which further stimulate the immune system. Human GM-CSF is currently being evaluated in the clinic for the treatment of neutropenia following chemotherapy and as an adjuvant in cancer therapy. The heterologous nucleotide sequence employed may comprise a heterologous gene, gene fragment or combination of genes.
The invention is also applicable to corrective gene therapy, to improve the target cell""s usefulness or viability. For example, normal CD34+ cells can be transduced with viral vector as described herein encoding a DNA repair enzyme such as 06-methylguanine DNA methyltransferase (MGMT), for protection of target cells e.g. during chemotherapy e.g. with nitrosourea, see T Moritz et al, Cancer Res 55(12) (1995) pp 2608-2614; R Maze et al, Proc Nat Acad Sci USA 93(1) 1996 206-210; or against radiation damage. Other genes for corrective gene therapy, of the kinds mentioned above, may also be transduced as transferred to target cells.
Heterologous DNA, e.g. further DNA, can usefully be introduced into the virus vector for other purposes, e.g. to encode expressibly an integrase such as one that is known to be able to act to integrate viral-vector DNA into the host genome so that the vector DNA becomes propagated when host cell mitosis occurs; and for other purposes.
Furthermore, according to embodiments of the invention, there are provided materials and methods to insert corrective or lethal genetic material to destroy or modulate malignant blast cells. This can be done for example by the expression in the target cell, by means of the herpesviral vector methods described herein, of antisense RNA or ribozyme sequences corresponding to genetic material encoded by the vector: for example as indicated in D Marcola et al, xe2x80x98Antisense Approaches to Cancer Gene Therapyxe2x80x99, Cancer Gene Ther 2 (1995) pp 47 et seq.
Techniques for use of antisense polynucleotides are known per se, and are readily adaptable to the specificity needed for the present application by using suitable nucleotide sequences, e.g. of at least about 12 nucleotides complementary in sequence to the sequence of a chosen target; by choosing from among known promoters suitable to the cellular environment in which they are to be effective, and other measures well known per se.
For example, techniques for use of antisense RNA to disrupt expression of a target gene are indicated (in connection with a sialidase gene) in specification WO 94/26908 (Genentech: TG Warner et al). Techniques for using antisense oligonucleotides capable of binding specifically to mRNA molecules are also indicated in specification. WO 94/29342 (La Jolla Cancer Research Foundation and the Regents of the University of Michigan: R Sawada et al) (in particular connection with mRNA encoding human lamp-derived polypeptides). Techniques for antisense oligonucleotides complementary to target RNA are indicated in specification WO 94/29444 (Department of Health and Human Services: B Ensoli and R Gallo) (as applied to basic fibroblast growth factor RNA). Techniques for using antisense oligonucleotides having a sequence substantially complementary to an mRNA which is in turn complementary to a target nucleic acid, in order to inhibit the function or expression of the target, are indicated in WO 94/24864 (General Hospital Corporation: HE Blum et al), (as applied to inhibition of hepatitis B viral replication). A review of antisense techniques is given by D Mercola and J S Cohen, ch.7 pp 77-89 in in R E Sobol and K J Scanlon (eds.) xe2x80x98Internet Book of Gene Therapy: Cancer Therapeuticsxe2x80x99 (Appleton and Lange, Stamford, Connecticut, 1995). Applications to other target specificities are readily accessible by adaptation.
Techniques for using ribozymes to disrupt gene expression are also known per se. For example, techniques for making and administering ribozymes (or antisense oligonucleotides) in order to cleave a target mRNA or otherwise disrupt the expression of a target gene are indicated in specification WO 94/13793 (Apollon: C J Pachuk et al) (as applied to ribozymes that target certain mRNAs relevant to leukemias). A review of ribozyme techniques is given in M Kashani-Sabet and K J Scanlon, ch. 8 pp 91-101 in RE Sobol and K J Scanlon (eds.) xe2x80x98Internet Book of Gene Therapy: Cancer Therapeuticsxe2x80x99 (Appleton and Lange, Stamford, Conn., 1995). Here also, applications to other target specificities are readily accessible by adaptation.
A lethal gene can also be inserted into the vector to destroy the transduced cell: for example a gene that is lethal in connection with an administered pharmaceutical, as described in e.g. specification WO 95/14100 (Wellcome Foundation: C Richards et al), exemplifying a gene encoding cytosine deaminase (CDA) under control of a CEA promoter, which when introduced into a cell is lethal in connection with administration of 5-fluorocytosine, transformed by the CDA into toxic 5-fluorouracil.
The recombinant herpesvirus used to carry a gene encoding an immunomodulatory protein or other genetic material as discussed herein, is preferably an attenuated and/or replication-defective herpesvirus.
The mutant herpesvirus can usefully be a mutant of any suitable herpesvirus; e.g. a non-transforming mutant of a mammalian herpesvirus; e.g. a mutant of a non-transforming human herpesvirus, especially for example a coated or enveloped mutant herpesvirus. Examples of herpesviruses of which mutants are provided and can be used as vectors according to embodiments of the invention include herpes simplex virus of type 1 (HSV-1) or type 2 (HSV-2), a human or animal cytomegalovirus (CMV), e.g. human cytomegalovirus (HCMV), varicella zoster virus (VZV), and/or human herpesvirus 6 and 7. EBV is less desirable, except in the form of a non-transforming mutant, because of its normally transforming properties. Animal viruses of which mutants are provided according to embodiments of the invention include pseudorabies virus (PRV), equine and bovine herpesvirus including EHV and BHV types such as IBRV, and Marek""s disease virus (MDV) and related viruses.
The nomenclature of the genes of herpesviruses and their many corresponding homologues is diverse, and where the context admits, mention of a gene in connection with a herpesvirus includes reference, in connection with other herpesviruses possessing a homologue of that gene, to the corresponding homologue.
Suitable herpesviruses to be used as a basis for recombination to produce a vector suitable for use according to the present invention include defective herpesviruses conforming with the general or specific directions in specification WO 92/05263 (Inglis et al: Immunology Limited) (the disclosure of which is incorporated herein by reference), which describes for example the use as an immunogen or vaccine of a mutant virus whose genome is defective in respect of a gene essential for the production of infectious virus, such that the virus can infect normal host cells and undergo replication and expression of viral antigen genes in such cells but cannot produce infectious virus. WO 92/05263 particularly describes an HSV virus which is disabled by the deletion of a gene encoding the essential glycoprotein H (gH) which is required for virus infectivity (A Forrester et al, J Virol 66 (1992) 341-348). In the absence of gH protein expression non-infectious virus particles providing almost the complete repertoire of viral proteins are produced. These progeny particles, however, are not able to infect host cells and spread of the virus within the host is prevented. Such a virus has been shown to be an effective immunogen and vaccine in animal model systems (Farrell et al, J Virol 68 (1994) 927-932; McLean et al, J Infect Dis, 170 (1994) 1100-9).
Such mutant viruses can be cultured in a cell line which expresses the gene product in respect of which the mutant virus is defective.
The literature also describes cell lines expressing proteins of herpes simplex virus: the gB glycoprotein (Cai et al, in J Virol 61 (1987) 714-721), the gD glycoprotein (Ligas and Johnson, in J Virol 62 (1988) 1486) and the Immediate Early protein ICP4 (Deluca et al, in J Virol 56 (1985) 558). These too have been shown capable of supporting replication of viruses inactivated in respect of the corresponding genes.
Complete or substantial sequence data has been published for several viruses such as human cytomegalovirus CMV (Weston and Barrell in J Mol Biol 192 (1986) 177-208), varicella zoster virus VZV (AJ Davison and Scott, in J Gen Virol 67 (1986) 759-816) and herpes simplex virus HSV (McGeoch et al, in J. Gen. Virol. 69 (1988) 1531-1574 and further references cited below). The gH glycoprotein is known to have homologues in CMV and VZV (Desai et al, in J Gen Virol 69 (1988) 1147).
Suitable examples of such genes are genes for essential viral glycoproteins, e.g. (late) essential viral glycoproteins such as gH, gL, gD, and/or gB, and other essential genes. Essential and other genes of human herpesviruses are identifiable from DJ McGeoch, xe2x80x98The Genomes of the Human Herpesvirusesxe2x80x99, in Ann Rev Microbiol 43 (1989) pp 235-265; DJ McGeoch et al, Nucl Acids Res 14 (1986) 1727-1745; D J McGeoch et al, J mol Biol 181 (1985) 1-13, for data and references cited therein. Reference is also made to data for homologues of gH glycoprotein in for example CMV and VZV, published e.g. in Desai et al, J Gen Virol 69 (1988) 1147).
Also useful as virus vectors in the present invention are for example the mutants such as HSV-1 mutant (in1814) unable to trans-induce immediate early gene expression, and essentially avirulent when injected into mice, described by C I Ace et al, J Virol 63(5) 1989 pp 2260-2269. Specification WO 91/02788 (CM Preston and CI Ace: University of Glasgow) describes useful HSV1 mutants including in 1814 capable of establishing latent infection in a neuronal host cell and of causing expression of an inserted therapeutic gene. Further examples of virus vectors useful in the invention are based on a mutation in a herpesvirus immediate early gene, e.g. a gene corresponding to ICP0, ICP4, ICP22 and ICP27. Mutations can be used in combination, e.g. as disclosed in WO 96/04395 (P Speck: Lynxvale), incorporated by reference. Also suitable as virus vectors for use in the present invention are such neuroattenuated HSV1 mutants as mutant 1716 (BP Randazzo et al, Virology 211 (1995) pp 94-101).
For herpesviruses reference is further made to data published for example in respect of human cytomegalovirus CMV (Weston and Barrell in J Mol Biol 192 (1986) 177-208), and varicella zoster virus VZV (AJ Davison et al, in J Gen Virol 67 (1986) 759-816).
According to certain examples of the present invention as described in further detail below, a genetically inactivated virus immunogen such as a vaccine provides an useful carrier for genes encoding immunomodulatory proteins. The virus vaccine can infect cells of the vaccinated host leading to intracellular synthesis of the immunomodulatory proteins. If the genetically inactivated vaccine is also acting as a vector for delivery of foreign antigens, then the immune response against the foreign antigen may be enhanced or altered.
Since these replication defective viruses can undergo only a single cycle of replication in cells of the vaccinated host, and fail to produce infectious new virus particles, production of the immunomodulatory proteins is confined to the site of vaccination, in contrast to the situation with a replication competent virus, where infection may spread. Furthermore, the overall amounts of immunomodulatory protein produced, though locally sufficient to stimulate a vigorous immune response, will be less than that produced by a replication competent virus, and less likely to produce adverse systemic responses.
In such a preferred embodiment, the heterologous nucleotide sequence, usually comprising a gene encoding immunomodulatory or other protein, is inserted into the genome of the mutant virus at the locus of the deleted essential gene, and most preferably, the heterologous nucleotide sequence completely replaces the gene which is deleted in its entirety. In this way, even if any unwanted recombination event takes place, and results in the reinsertion of the deleted gene from a wild source into the mutant virus, it would be most likely to eliminate the inserted heterologous nucleotide sequence. This would avoid the possibility that a replication competent viral carrier for the heterologous nucleotide sequence would be produced. Such a recombination event would be extremely rare, but in this embodiment, the harmful effects of such an occurrence would be minimised.
Materials and methods according to the invention can be used to evoke immunological effector mechanisms activated by cellular immunogens such as therapeutic vaccines, in particular to evoke specific cytotoxic T lymphocytes (CTLs) directed against target antigens. Such CTLs can exert a beneficial effect by tending to recognise and destroy tumor cells, and can also be used ex-vivo in a variety of diagnostic and/or therapeutic methods.
Where there are antigenic differences between tumor cells and normal cells, they can be recognised by the immune system, provided that the tumor-specific antigens are available in the correct form to stimulate an immune response. This avoids the need to identify tumor specific markers.
CTLs destroy cells on the basis of antigen recognition in conjunction with host major histocompatibility complex (MHC) antigens; peptides generated from the antigenic target within the cytoplasm of the host cell form a complex with host MHC molecules and are transported to the cell surface, where they can be recognised by receptors on the surface of CTLs.
One method of using the vectors, provided by this invention, is therefore to prepare a cellular immunogen such as a vaccine from tumor material derived from one or more individuals and to administer this as an immunogen or vaccine for treatment of other subjects, e.g. patients. If a CTL response against the tumor cells is desired, however, for the reasons outlined above, the target antigens should be presented in the context of the correct MHC molecules. An immunogen or vaccine prepared from a tumor of one individual may not always therefore be appropriate for another individual with a different MHC type. Since MHC molecules vary from individual to individual, it is generally necessary, in order to activate CTL responses against the target antigens, to present the relevant target antigen to the immune system in the correct MHC context. Thus for use as an immunogen such as a therapeutic vaccine, in general it is considered that the selected target antigen is best introduced into the treated subject""s or patient""s own cells in order to generate an appropriate CTL response.
It can therefore be especially useful to base the tumor immunogen or vaccine on a patient""s own tumor cells, a procedure known as autologous vaccination. A further major advantage of this way of use is that it can take advantage of antigenic targets that may be unique to a particular tumor; it is considered that the deregulated cell cycle control that is the basis of tumor growth can, over a period of time, lead to the accumulation of genetic changes manifested as new antigenic determinants. In this connection, the last-mentioned embodiments of the present invention can avoid or solve a problem with autologous vaccination procedure, namely that autologous tumor cells are poorly immunogenic.
Procedures according to examples of the invention can involve introduction of a target gene into tumor cells removed from a subject, by laboratory procedures after which the cells so treated are reintroduced into a subject to be treated (ex-vivo treatment). An alternative procedure according to certain examples of the invention is to introduce the target gene directly into tumor cells of the patient (in vivo treatment). The advantage of an in-vivo procedure is that no laboratory manipulation of the patient""s tumor cells is required. A drawback can be that effective gene transduction may be more difficult to achieve in vivo, or more difficult to achieve to a desired degree. Other, non-tumor, cells can also usefully be transfected with the virus vectors.
In a particular example, the recombinant herpesvirus is based on a disabled form of the herpes simplex virus carrying a deletion in the glycoprotein H (gH) gene, a protein present on the surface of the virus particle that is involved in entry of virus into susceptible cells. This virus can only be replicated in a producer cell line that complements the essential function missing in the viral genome: a useful example of a recombinant complementing cell line is one which has been engineered to express stably the same HSV gH gene as was deleted from the virus vector. The virus generated from the producer cell line acquires the cell-encoded gH gene product as part of its structure and is infectious. This virus preparation can infect normal cells in the same manner as wild type virus. Once in the cell, the virus genome can be intracellularly replicated, and genes carried by the genome can be expressed as protein. However the absence of a functional gH protein when the defective virus infects a normal cell results in failure to generate new infectious virus particles. The gH-deleted virus is considered to be safe to administer as a vaccine or a gene delivery vehicle.
It is preferable that a vector such as a HSV vector for cancer immunotherapy is fully disabled and unable to spread within the treated host. A useful vector can, however, be based on any HSV virus that is deemed sufficiently safe to be used in a clinical setting. It is also preferable that heterologous genes incorporated into such a gH-deleted HSV genome are inserted at the locus from which the gH gene was removed, to minimise the risk of transfer of the heterologous gene by homologous recombination to wild type HSV that might co-exist in the treated individual. The heterologous gene can however instead be inserted at any site within the virus genome.
A further adaptation of the method within the scope of the invention is to deliver the appropriate genetic material, e.g. a gene encoding an immunomodulatory protein, in the form of herpesviral amplicons packaged within herpesviral particles. Amplicon DNA is DNA that contains an origin of replication of a herpesviral genome together with DNA sequences that can direct packaging of this DNA into virus particles. Where such amplicons are present in cells along with corresponding herpesvirus (helper virus), expression of amplicon DNA can occur along with expression of herpesviral DNA. Foreign genes can be cloned into such amplicons and thus expressed in cells infected with the amplicons as well as with herpesvirus. Particles containing packaged amplicons can be phenotypically equivalent to the corresponding helper virus and hence able to infect the same host cell and are considered herein as among the defective mutant herpesvirus suitable as vectors for use in the practice of the invention. Thus virally-packaged amplicons can also be used to deliver selected DNA to desired cells. Amplicons and processes for their preparation that can be used or readily adapted for use in examples of the performance of this invention, along with further details, are described in further detail in WO 96/29421 (Efstathiou et al: Cantab Pharmaceuticals Research Ltd and Cambridge University Technical Services Ltd).
It is preferable that the HSV helper virus used for packaging the amplicons is, by itself, not harmful to the host, and so a disabled virus with an essential gene deleted, such as the gH-deleted virus described above provides an ideal helper virus as described in WO 92/05263 and other related references cited herein. Other useful helper viruses can, however, be based on herpesvirus sufficiently attenuated or disabled to be used in a clinical setting, not necessarily one that is entirely replication-defective.
The invention described here can be used to deliver chosen genetic material, e.g. DNA encoding a chosen protein such as an immunomodulatory protein, to tumor cells for the purposes of therapy. The range of genes that can be delivered for the purpose of stimulating an immune response includes genes for cytokines, immunostimulators, lymphotactin, CD40, OX40, OX40 ligand, and other genetic material mentioned herein, which can be included in the vector as single genes or multiple genes, or multiple copies of one or more genes.
In embodiments of the present invention, for example using vectors and target cells as particularly described herein below, normal and malignant human hemopoietic progenitor cells can be rapidly transduced with efficiencies ranging from 60% to 100%; the levels of transduction and gene expression that have been achieved are considered to represent high efficiency, particularly for these targets.
Embodiments of the present invention can also produce useful rapidity of expression of a transferred gene. For example under conditions as specifically described herein, positivity for expression of the transferred gene has been obtained in 80% to 100% of CD34+ cells as well as AML and ALL blasts within 24 hours after exposure to the vector. It has also been found that embodiments of the invention can provide a preparation of transduced cells that produce, and for example release, the product of the transferred gene for at least 7 days at a level proportional to the MOI (multiplicity of infection, usually reckoned in plaque-forming units (pfu)lcell), for example at MOI in the range 0.05-20, e.g. in the case of GM-CSF produced in human primary leukaemic cells by expression of the corresponding gene transferred by a gH-deletant herpesviral vector.
Accordingly, it is seen that embodiments of the present invention enable the production of immunogens, e.g. human leukaemia immunogens, in cases where the production of corresponding immunogens has presented logistic problems up to now. (Although in the case of leukaemic blasts, for example, it might be or become possible to obtain high levels of cytokine production with retroviral or adenoviral vectors in certain susceptible examples of cells, embodiments of the present invention have been found to enable consistently achievable useful high proportions of leukaemic blasts to be transduced from all patients so far tested, thus presenting useful advantage in clinical work.)
The present invention is further described below by the help of examples of procedures and products and of parts of procedures and products given by way of example only and not of limitation.
The construction of suitable vectors is illustrated non-limitatively by reference to the accompanying drawings, in which: